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Peter Jackstadt1, Horst Zahner2 and Gerd Hobom1, 1 Institut fr Mikrobiologie und Molekularbiologie der Justus-Liebig Universitt Gieen, Frankfurter Str. 107, D-35392, Gieen, 2 Institut fr Parasitologie der Justus-Liebig-Universitt Gieen, Rudolf-Buchheim-Str. 2, D-35392 Gieen
Introduction
The Biolistic DNA transfer technique was first established for plant
cells and plant tissues (Sandford et al., 1987; Jefferson et al., 1987;
Bruce et al., 1989; Armaleo et al., 1990). More recent applications have
been DNA transfer experiments aiming at DNA vaccination of animals (genetic
immunization; Sundaram et al., 1996), for example a bombardment of mice
to generate protective antibodies, instead of the DNA injection used for
this purpose in the past.
Here we report on the potential to use this convenient method for transfection of nematodes, not only for the free-living soil nematode Caenorhabditis elegans, the well known model organism in its class, but also for the cotton rat filaria Litomosoides sigmodontis, which serves as model for human pathogenic filariae (Zahner, Hobom and Stirm, 1995), such as Wuchereria bancrofti or Brugia spp. These parasitic species cause a severe lymphatic disease called filariasis (elephantiasis), from which more than 100 million human beings suffer worldwide.
Methods
PREPARATION OF ADULT HERMAPHRODITE C. ELEGANS AND COTTON RAT PARASITE
L. SIGMODONTIS FOR MICROPARTICLE BOMBARDMENT
C. elegans is cultivated on NGM agar plates (Brenner, 1974). Wild-type
worms (strain N2 var. Bristol) are fed on Escherichia coli OP-50 (exogenous
uracil dependent mutant) seeded on those plates. The surface of a densely
populated NGM agar plate is subsequently rinsed with tap water. A drop
of water containing nematodes is placed in the center of a 6 cm Petri
dish on ice (to reduce their motility). In this state C. elegans could
be used for microparticle bombardment (Figure 1A).
For the same purpose adult females of L. sigmodontis must be isolated from the pleural cavity of their laboratory host Mastomys coucha (Wegerhof and Zahner, 1985), and stored in RPMI medium (supplemented with 10% FCS, Gibco-BRL; 1% Penicillin and Streptomycin). Single parasites are transferred to an agar plate and used for microprojectile bombardment.
CONDITIONS OF BOMBARDMENT
DNA-coated gold particles are chosen as microparticles following a modified
Bio-Rad protocol for the Helios gene gun. The protocol is as follows:
1. Mix 50 mg of gold microparticles (Bio-Rad; a 1:1 mixture of particles
0.6 /1.0 in diameter) with 100 l of 0.05 M spermidine (Sigma), and
sonicate 3 times for 3 seconds in an Eppendorf tube (1.5 ml).
2. Add 200 l of purified supercoiled plasmid DNA to the carrier suspension (in a total volume of 100 l). It is possible to use more than one plasmid simultaneously.
3. CaCl2 (100 l, 1 M) is now added dropwise with continuous vortexing. Precipitation was allowed to take place for 10 minutes at room temperature.
4. Wash the suspension three times with 1 ml of absolute ethanol (Sigma-Aldrich), and pellet the sample by centrifugation (12,000 rpm for 1 minute, Heraeus Biofuge).
5. Finally, the pellet is resuspended in 3.5 ml of ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP; MW 360,000). In this state a DNA-coated gold particle suspension can be stored for up to 2 months at -20 C (if not directly used for cartridge preparation).
USING THE TUBING PREP STATION
1. Clean the inner surface of the Tefzel tubing (external size 10 mm;
internal diameter 6 mm; Bio-Rad) with 5 ml of absolute ethanol and dry
with nitrogen (grade 4.8; 99.998% pure) in the rotating tubing holder
for at least 5 minutes.
2. Sonicate the DNA-coated particle suspension in ethanol for 3 seconds (to achieve a homogeneous solution) and load it into the tubing at a rate of 5.56 ml/min using a syringe.
3. Allow the DNA-coated particles to settle for 3 minutes.
4. Remove the ethanol solution quickly (within 30 seconds).
5. For drying the wall-attached particles, flush nitrogen gas through the tubing for 3 minutes at a flow rate of 0.35 l/min.
6. Finally, cut the resulting particle-covered tubing into individual cartridges 16 mm in length (one full length of tubing results in nearly 50 cartridges) to fit into the 12-shot barrels of the Helios gene gun (Bio-Rad). See Figure 2.
7. In a desiccated atmosphere, these can be stored at 4 C for up to 8 months without losing activity.
8. Deliver the DNA-coated gold particles using 4 blasts of helium (grade 4.5; 99.995% pure) applied to the cartridges at a pressure of 300 p.s.i. and a distance of 3 cm to the target (in the case of C. elegans).
9. A pressure of 120180 p.s.i. at a distance of 2 cm using 1 blast of helium is useful for the transfer into parasitic nematode L. sigmodontis. After this procedure, the female parasites may be cultivated at 37 C for up to 2 weeks in RPMI medium (5% CO2).
SELECTION FOR TRANSFORMED TRAITS OF C. ELEGANS
While the majority (up to 90%) of C. elegans hermaphrodites are killed
during Biolistic DNA transfer, the survivors with their gold particles
incorporated may be cultivated on new E. coli-seeded NGM agar plates (Figure
2A shows particles within a living hermaphrodite). F1/F2 progeny nematodes
were screened 3 to 4 days later for visible dominant mutant marker rol-6
(Kramer et al., 1990 ). Animals carrying roller allele su-1006 exhibit
a helically twisted cuticle and body. Muscle contractions, which would
normally propel the worm forward or backward in a sweeping sinusoidal
motion, will cause the nematode instead to roll over its longitudinal
axis and to move in circles (Mello et al., 1991; Fire, 1986; Kramer et al., 1990). In the case of L. sigmodontis, no special selection mode is
necessary because the success of bombardment can be visualized by standard
light microscopy to make visible the gold particles now located within
these filariae (see Figure 2B).
Results
The established mode of DNA transfer into C. elegans is microinjection
of DNA into the gonadal syncytium (Stinchcomb et al., 1985; Fire, 1986).
Under standard selection conditions promoter-reporter gene cDNA constructs
are used, in co-transfection with a visible dominant such as mutation
rol-6 (su1006), which leads to roller movement phenotype of transformed
worms (already in F1 generation) and is easily detectable within a background
of normally moving animals. The same transfection efficiency has been
observed for this Biolistic DNA transfer method as for microinjection
experiments (Fire, 1986), while this new strategy is less time consuming
and much more convenient for co-transfection experiments. In the F1 generation,
DNA transfer rates of up to 1% were observed using this selection method,
which became considerably enriched during following generations after
further transfer (F3/F4: 20%; F5/F6 up to 90%), proving their genetic
fixation.
Besides marker gene rol-6, we have employed two different reporter genes, a mutant form of GFP (green fluorescent protein), which was originally cloned from the jellyfish Aequorea victoria (Prasher et al., 1992; Cody et al., 1993; Chalfie et al., 1994) with enhanced fluorescence (Siemering et al., 1996), and -galactosidase from Escherichia coli. Expression was visualized by standard fluorescence microscopy (GFP; see Figure 4) or by histochemical staining (lacZ).
In both cases we have observed marker gene expression under transcriptional control of the SV40 early promoter (Figure 3 A and B), or of the C. elegans actin-1 core promoter after Helios gene gun mediated delivery of Au/DNA particles into the nematode. The lacZ expression of constructs containing the actin-1 core promoter of C. elegans (Files et al., 1983; Krause et al., 1989; Stone and Shaw, 1993) in DNA transfected L. sigmodontis will even be analyzed histochemically.
Discussion
The Biolistic DNA-transfection procedure, because of its simple mode of
handling, has an important potential in various innovative molecular biology
applications, such as DNA vaccination, protein targeting, and basic research
applications such as gene regulation studies. The new Biolistic technology
should therefore be able to substitute for other applications in this
field. Also, the prepared DNA/Au particles can be stored for rather long
periods, at least up to 8 months, which makes repetitions or comparative
experiments more meaningful. In applying this technique to various nematodes,
it is conveniently possible to use a couple of hundred target worms of
C. elegans and proceed in comparatively low precision, while DNA injections
have to be done individually and precisely into the gonads. Parasitic
worms, however, can only be transfected on an individual basis or in small
groups by Biolistic gene transfer. In all experiments designed, reporter
gene expression is observed independent from distance of initial gold
particle target sites, clearly indicating an DNA drift within the entire
bodies. Altogether, this technique is a reproducible and efficient method
of producing fertile transgenic nematodes.
References
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) via
Sonderforschungsbereich 535. We thank Noelia Pohl, Ulrike Ruppert and
Brigitte Hofmann for their exceptional technical assistance. Many thanks
to PD Dr. R. Dennis for helpful discussion and comments on the manuscript.
Biolistic is a registered trademark of E. I. duPont de Nemours and Co.
Biolistic technology is exclusively licensed to Bio-Rad Laboratories.
Eppendorf is a trademark of Eppendorf Geratebau Netheler + Hinz, GmbH.
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